The present invention concerns compounds with anti-viral activity, including novel inhibitors of the 3C, 2A and 3C-like cysteine proteases or picornaviridae and related viruses (see Gorbalenya, Prospective Drug Discovery Design, 6: 64-86, 1996).
The picornavirus family encompasses single-stranded positive-sense RNA viruses, encapsulated in a protein capsid (Reviewed in Rucekert, 1996, i Fields, Virology, 609-654). These viruses cause a wide range of diseases in humans and other mammals, including the common cold (rhinoviruses), gastro-intestinal ailments (enteroviruses, such as coxsackievirus and echovirus), poliomyelitis (the enterovirus poliovirus), heart diseases (cardioviruses), hepatitis (hepatitis A) and foot and mouth disease (aphtoviruses). Rhinoviral common cold is of specifically noted medical and economical significance, due both to is ubiquity (the major cause of acute illness in the United States) and to the debilitating effect of the disease and consequential loss of work days (see McKinlay, Ann. Rev. Microbiol., 46:635-54, 1992). Related viral families of the picornavirus supergroup, such as flaviviridae and potyviridae, cause various diseases in agriculturally significant crops, such as potatoes (see Ryan, J. Gen. Virol., 4:699-723, 1997).
After infection of the host cell, the picornaviral RNA is translated into a single polyprotein which has auto-catalytic proteolytic activity, cleaving itself into mature viral proteins. The 2A and 3C proteolytic enzymes, which are part of the picornaviral autocatalytic polyproteins, are responsible for these cleavages. The 2A protease cleaves co-translationally between the structural and non-structural protein precursors and the 3C protease cleaves most of the remaining sites post-translationally.
The 3C and in some cases 2A, proteases are responsible for maturation of the picornavirus and are crucial for completion of the picornaviral life cycle. As a result, the 3C protease has been a prime target for existence structural and mechanistic investigations during the last few years, and its mechanism of action and structural features has been determined (Kreisberg et al, Organic Reactivity: Physical and Biological Aspects, 110-122 (1995)).
The 3C and 2A proteases have somewhat different specificities for substrates. Furthermore, the two proteases have different mechanisms of action because the 2A protease requires zinc, while the 3C protease does not.
Attempts to find treatments for diseases caused by a picornavirus species have been largely directed towards finding inhibitors for the 3C protease, since such inhibition would prevent production of new virions as there are no native cellular proteases which can replace the cleavage activity of these viral proteases. Therefore, finding an efficient inhibitor of 3C picornavirus protease activity, or of other viral activity, would be valuable for the treatment of a large number of viral diseases, both in humans and in lower animals.
The first inhibitor of the 3C protease to be found by screening was thysanone, an antibiotic compound obtained from Thysanphora penicilidies (Singh et al, Tetrahedron Lett., 32: 5279-82 (1991)). However, this compound was not developed into a pharmaceutical composition, since it exhibited modest protease inhibitory activity and was found to be an inhibitor of the elastase enzyme present in erythrocytes.
Two additional antibiotic compounds, of fungal origin, termed citrinin hydrate and radicinin were obtained by screening over 20,000 microbial extracts (Kadam et al. J. Antibiotics 7: 836-839 (1994)). These two compounds were weaker inhibitors of picornaviral proteases than thysanon. In this same screening process, a new compound termed kalafungin, which is also an antibiotic compound, was discovered by structural comparison to radicinin. Kalafungin was found to be a better inhibitor, by three orders of magnitude, than radicionin an citrinin hydrate (McCall et al, Biotechnology, 12: 1012-1016 (1994)). However, none of these inhibitors has yielded a clinically useful compound, combining high anti-viral activity but low toxicity.
Another group of inhibitors, substituted isatins, has also been examined (S. E. Webber et al., Med. Chem., 39:5072-5082, 1996). Certain members of this group show significant inhibition of 3C proteases with concentrations in the nmolar range, but are highly toxic. Other members of the group are relatively non-toxic, but have poor anti-viral activity. Thus, none of the background art compounds combine strong protease inhibitor or anti-viral effects with low toxicity.
There is therefore an unmet medical need for new compounds which have strong anti-viral activity against the picornaviruses.